Towards the development of artificial endocrine tissues: (31)P NMR spectroscopic studies of immunoisolated, insulin-secreting AtT-20 cells

Transformed, insulin-secreting endocrine cells have been proposed as an alternative to islets for the development of a bioartificiat pancreas. With appropriate immunoprotection, such cells may be implanted without the need for patient immunosuppression. Use of continuous cell lines alleviates the cell availability limitation, but poses questions regarding thestability and biochemical and secretory function of the preparation, especially in the longterm. We have developed a bioreactor/perfusion system, compatible with a horizontal-bore NMR instrument, that can maintain immunoprotected endocrine cells for prolonged periods of time. (31)P NMR spectroscopy was used to study the bioenergetics of recombinant, insulin-secreting mouse pituitary AtT-20 cells entrapped as spheroids in calcium alginate/poly-L-lysine/alginate beads. NMR provided data verifying the macroscopic homogeneity within the bioreactor and allowing the evaluation of changes in cellular bioenergetics for a period of 70 days under different culture conditions. Levels of high-energy phosphates changed slightly during the first 40 days of the experiment, then decreased considerably as cell death occurred. Rates of glucose consumption and insulin-related peptide secretion also remained constant for 40 days and decreased rapidly thereafter. This study constitutes the beginning of an extensive quantitative analysis of the biochemistry of transformed endocrine cell lines in a sequestered, artificial tissue environment. (c) 1995 John Wiley & Sons Inc.     

Development of a bioartificial pancreas: I. long-term propagation and basal and induced secretion from entrapped betaTC3 cell cultures

Bioartificial pancreatic constructs based on immunoisolated, insulin-secreting cells have the potential for providing effective, long-term treatment of type I (insulin-dependent) diabetes. Use of insulinoma cells, which can be amplified in culture, relaxes the tissue availability limitation that exists with normal pancreatic islet transplantations. We have adopted mouse insulinoma betaTC3 cells entrapped in calcium alginate/poly-L-lysine/alginate (APA) beads as our model system for a bioartificial pancreas, and we have characterized the effects of long-term propagation and of glucose concentration step changes on the bioenergetic status and on the metabolic and secretory activities of the entrapped cells. Cell bioenergetics were evaluated nonivasively by phosphorus-31 nuclear magnetic resonance ((31)P NMR) spectroscopy, and metabolic and secretory parameters by assaying cell culture medium. Data indicate that net cell growth occurred between days 3 and 10 of the experiment, resulting in an approximate doubling of the overall metabolic and secretory rates and of the intracellular metabolite levels. Concurrently, a reorganization of cell distribution within the beads was observed. Following this growth period, the measured metabolic and secretory parameters remained constant with time. During glucose step changes in the perfusion medium from a high concentration of 12 to 15 mM to 0 mM for 4.5 h to the same high glucose concentration, the oxygen consumption rate was not affected, whereas insulin secretion was always glucose-responsive. Intracellular nucleotide triphosphates did not change during 0 mM glucose episodes performed early in culture history, but they declined by 20% during episodes performed later in the experiment. It is concluded that the system of APA-entrapped betaTC3 cells exhibits several of the desirable characteristics of a bioartificial pancreas device, and that a correlation between ATP and the rate of insulin secretion from betaTC3 cells exists for only a domain of culture conditions. These findings have significant implications in tissue engineering a long-term functional bioartificial endocrine pancreas, in developing noninvasive methods for assessing construct function postimplantation, and in the biochemical processes associated with insulin secretion.     

Performance of an external loop air-lift bioreactor for the production of monoclonal antibodies by immobilized hybridoma cells

Summary The performance of an external loop air-lift bioreactor was investigated by assessing the inter-relationships between various hydrodynamic properties and mass transfer. The feasibility of using this bioreactor for the production of monoclonal antibodies by mouse hybridoma cells immobilized in calcium alginate gel beads and alginate/poly-l-lysine microcapsules was also examined. When the superficial gas velocity, V _g , in the 300 ml reactor was varied from 2 to 36 cm/min, the average liquid velocity increased from 3 to 14 cm/sec, the gas hold-up rose from 0.2 to 3.0%, and the oxygen mass transfer coefficient, k _L a, increased from 2.5 to 18.1 h^-1. A minimum liquid velocity of 4 cm/s was required to maintain alginate gel beads (1000 m diameter, occupying 3% of reactor volume) in suspension. Batch culture of hybridoma cells immobilized in alginate beads followed logarithmic growth, reaching a concentration of 4×10⁷ cells/ml beads after 11 days. Significant antibody production did not occur until day 9 into the culture, reaching a value of 100 g/ml of medium at day 11. On the other hand, bioreactor studies with encapsulated hybridoma cells gave monoclonal antibody concentrations of up to 800 g/ml capsules (the antibody being retained within the semipermeable capsule) and maximum cell densities of 2×10⁸ cells/ml capsule at day 11. The volumetric productivities of the alginate gel immobilized cell system and the encapsulated cell system were 9 and 3 g antibody per ml of reactor volume per day, respectively. The main advantage of the bioreactor system is its simple design, since no mechanical input is required to vary the hydrodynamic properties.     

Immobilization of Saccharomyces uvarum cells in porous beads of polyacrylamide gel for ethanolic fermentation

Summary A procedure which does not involve the use of an immiscible organic solvent phase is described for the entrapment of yeast cells in porous beads of polyacrylamide gel. The cells are rapidly dispersed at 4° C in an aqueous solution containing sodium alginate and acrylamide-N,Nmethylene-bis-acrylamide monomer, and the suspension is immediately dropped into a solution of calcium formate to give calcium alginate coated beads. Polyacrylamide gel forms within the bead. The calcium alginate is subsequently leached out of the composite bead with either sodium citrate or potassium phosphate buffer solution. Cells of Saccharomyces uvarum ATCC 26 602 entrapped in such polyacrylamide beads ferment cane molasses in batch mode at higher specific ethanol productivity than a free cell suspension. Their volumetric productivity in continuous fermentation is higher than that of Ca²⁺-alginate immobilized cells.NCL Communication No. 4383     

Hydantoinase activity of immobilized non-growing Pseudomonas putida cells

Summary Hydantoinase (dihydropyrimidinase E.C. 3.5.2.2) activity of Pseudomonas putida DSM 84 was evaluated using cells immobilized in alginate beads and in a microporous hollow fibre bioreactor. Conversion of dihydrouracil into N-carbamyl--alanine was most efficient with alginate-immobilized cells. A 40 to 45% conversion was obtained in shake flasks and in continuous mode with packed bed columns. The highest volumetric productivity was obtained with a packed bed column operated at a dilution rate of 0.5 h^-1 (99 g of product. 100 l^-1 per hour). After 96 h the alginate beads began to swell and break apart; no free cells were detected however. Despite some initial loss of cells from the microporous hollow fibre bioreactor, a steady state was later established and maintained for 400 h at dilution rates of 0.1 and 0.25 h^-1.     

Hybridoma cells in a protein-free medium within a composite gel perfusion bioreactor

A composite gel system has been developed combining the chemical and physical properties of calcium alginate and agarose gels. The results of growing composite gel immobilized hybridoma SPO1 cells in a protein-free medium within a fluidized-bed perfusion bioreactor are presented in this paper. During the continuous operation of this system, the total cell density reached 3.9×10⁷ cells per ml of beads (viability 79.6%). The specific productivity of monoclonal antibody of the immobilized hybridoma cells reached more than 1.5 g per 10⁶ viable cells per hour, compared with 0.5 for non-immobilized viable cells grown in a one liter agitated bioreactor with the same medium. Significant increases in cell metabolic activities, including substrate utilization and byproduct formation, were also observed. Leaching of materials from the beads was evident and the major fraction of released materials was alginate.     

Evaluation of Metabolomic Changes as a Biomarker of Chondrogenic Differentiation in 3D-cultured Human Mesenchymal Stem Cells Using Proton (1H) Nuclear Magnetic Resonance Spectroscopy

Purpose

The purpose of this study was to evaluate the metabolomic changes in 3D-cultured human mesenchymal stem cells (hMSCs) in alginate beads, so as to identify biomarkers during chondrogenesis using ¹H nuclear magnetic resonance (NMR) spectroscopy.

Materials and Methods

hMSCs (2×10⁶ cells/mL) were seeded into alginate beads, and chondrogenesis was allowed to progress for 15 days. NMR spectra of the chondrogenic hMSCs were obtained at 4, 7, 11, and 15 days using a 14.1-T (600-MHz) NMR with the water suppression sequence, zgpr. Real-Time polymerase chain reaction (PCR) was performed to confirm that that the hMSCs differentiated into chondrocytes and to analyze the metabolomic changes indicated by the NMR spectra.

Results

During chondrogenesis, changes were detected in several metabolomes as hMSC chondrogenesis biomarkers, e.g., fatty acids, alanine, glutamate, and phosphocholine. The metabolomic changes were compared with the Real-Time PCR results, and significant differences were determined using statistical analysis. We found that changes in metabolomes were closely related to biological reactions that occurred during the chondrogenesis of hMSCs.

Conclusions

In this study, we confirm that metabolomic changes detected by ¹H-NMR spectroscopy during chondrogenic differentiation of 3D-cultured hMSCs in alginate beads can be considered as biomarkers of stem cell differentiation.     

Immobilization of the methanogenic bacterium Methanosarcina barkeri

Whole cells of the methanogen Methanosarcina barkeri were immobilized in an alginate network which was crosslinked with Ca²⁺ ions. The rates of methanol conversion to methane of entrapped cells were found to be in the same range as the corresponding rates of free cells. Furthermore, immobilized cells were active for a longer period than free cells. The particle size of the spherical alginate beads (1.2 mm-3.7 mm ?) and thus diffusion had no obvious influence on the turnover of methanol. The half-value period for methanol conversion activity determined in a buffer medium was approximately 4 days at 37°C for entrapped cells. The apparent K_m value K for such cells was nearly 140mM and the V_max value was about 1.2 μmol methanol/min/mg entrapped protein. Therefore the high rates of methanol degradation measured, e.g., 0.5 μmol methanol/min/mg entrapped protein, indicated that the immobilization technique preserved the cellular functions of this methanogenic bacterium.     

Demonstration of multiple forms of bovine brain myristoyl CoA: protein N-myristoyl transferase

Pancreatic -cells are known to respond to hyposmolar stress by releasing insulin. It was evident from perifusion studies using islet cells from oblob-mice mixed with polyacrylamide beads that a similar type of secretory response can be obtained by isosmolar addition of 10–25 mM of the rapidly penetrating urea molecule. There was no effect with hyperosmolar addition of urea. The urea-induced insulin release differed from the ordinary stimulation of secretion in not disappearing but being more pronounced after previous heating to 45°C or removal of extracellular Ca²⁺. Isosmolar urea was exceptional as an insulin secretagogue in being effective also in the presence of the ₂-adrenergic agonist clonidine or when lowering the temperature to 24°C. Further support for the idea that isosmolar addition of rapidly penetrating molecules induces insulin release was obtained by test ing non-metabolizable glucose analogues. Whereas 25 mM 3-O-methyl-D-glucose doubled the secretory rate within 4 min, the non-permeant L-glucose had only a slight initial action. When not compensating for the alterations of the medium osmolarity 3-O-methyl-D-glucose was without effect. Although expansion of -cells cannot explain the existence of a pronounced initial secretory response to D-glucose it may under certain conditions contribute to the stimulatory effects of the sugar.     

Glucose-induced reduction of the sodium content in β-cell-rich pancreatic islets

The sodium contents of -cell-rich pancreatic islets fromob/ob-mice were measured with an integrating flame photometer. After washing to an apparent steady state with different types of ice-cold media, islets incubated in the absence of glucose contained 79–108 mmol sodium kg^–1 dry weight. Exposure to glucose resulted in 25 % reduction of the islet content of sodium. This effect became manifest in the presence of 5 mM glucose, there being no additional reduction with a further increase of glucose to 20 mM. Depression of Na⁺ activity may partially explain why glucose, under certain conditions, can lower cytoplasmic Ca²⁺ and even inhibit insulin release.     

Effects of prolonged maternal fast on the pancreas of 18–21-day-old foetal rats

Summary After maternal fasting for 72 h the pancreatic cells of 18-day-old foetal rats show a conspicuous enrichment in secretory material, with an increase of pancreatic insulin concentration and a marked development of the rough endoplasmic reticulum and the Golgi apparatus.The morphometric analysis shows that the intracytoplasmic migration of the secretory granules is inhibited, principally inside the cell web. Consequently the number of secretory granules fused with plasma membrane decreases and this is associated with a decreased foetal plasma insulin.The difference in the ultrastructural aspect of the cells of foetuses from fasting mothers and of foetuses from fed mothers is less conspicuous at 19 days of gestation and progressively disappears at 20 and 21 days.The modifications in ultrastructural aspect and in functional state are discussed.     

Immobilization of microsomes into alginate beads is a convenient method for producing glucuronides from drugs

Summary The production of glucuronides from drugs by immobilized microsomal uridine diphosphate (UDP)-glucuronosyltransferase has been investigated. Of all the immobilization methods used (covalent binding, adsorption by ionic or hydrophobic interactions), only entrapment of microsomes into alginate beads in the presence of polyethyleneimine was effective in producing high glucuronidation rates, thus leading to the formation of large amounts of metabolites. The performance of the bioreactor was optimized with the drug 3-azido-3-deoxythymidine (AZT), active against the human immunodeficiency virus, as a model substrate of UDP-glucuronosyltransferase. Calcium (12 mm) could optimally improve the stability of microsomes entrapped in alginate beads. Upon immobilization, enzyme activation occurred, leading to a fivefold increase in specific activity. The determination of apparent K _m and V _max revealed that AZT was a better substrate for the immobilized enzyme than free microsomes. The AZT-glucuronide production obtained after 6 h was threefold higher than that observed with free microsomes. This bioreactor was also efficient in production of glucuronides from structurally different compounds such as bilirubin, 4-nitrophenol, clofibric acid, pirprofen, dextrorphan or morphine, the corresponding glucuronide of which possesses pharmacological or toxicological interest. Offprint requests to: J. Magdalou     

Integrated immobilized cell reactor-adsorption system for β-cyclodextrin production: a model study using PVA-cryogel entrapped Bacillus agaradhaerens cells

Production of cyclodextrins (CDs) by immobilized cells of the alkaliphilic Bacillus agaradhaerens LS-3C with integrated product recovery was studied. The microorganism was entrapped in polyvinyl alcohol-cryogel beads and used as a convenient source of immobilized cyclodextrin glycosyltransferase (CGTase). On activation by incubation in the cultivation medium containing 1% (w/v) starch, the entrapped cells multiplied and secreted CGTase with an activity of 2–3 mg -cyclodextrin h^–1 g^–1 beads. The immobilized biocatalyst exhibited maximum activity at pH 9 and 50 °C, and formed cyclodextrins comprising 92–94% -CD and remaining -CD. The cyclodextrin product from the immobilized cell bioreactor was continuously recovered by adsorption to Amberlite XAD-4 in a recycle batch mode. The product adsorption was facilitated at low temperature while hot water was used for elution.     

Structure–Function Relationship of New Crotamine Isoform from the <Emphasis Type="Italic">Crotalus durissus cascavella</Emphasis>

In this work we isolated a novel crotamine like protein from the Crotalus durissus cascavella venom by combination of molecular exclusion and analytical reverse phase HPLC. Its primary structure was:YKRCHKKGGHCFPKEKICLPPSSDLGKMDCRWKRK-CCKKGS GK. This protein showed a molecular mass of 4892.89 Da that was determined by Matrix Assisted Laser Desorption Ionization Time-of-flight (MALDI-TOF) mass spectrometry. The approximately pI value of this protein was determined in 9.9 by two-dimensional electrophoresis. This crotamine-like protein isolated here and that named as Cro 2 produced skeletal muscle spasm and spastic paralysis in mice similarly to other crotamines like proteins. Cro 2 did not modify the insulin secretion at low glucose concentration (2.8 and 5.6 mM), but at high glucose concentration (16.7 mM) we observed an insulin secretion increasing of 2.7–3.0-fold than to control. The Na⁺ channel antagonist tetrodoxin (6 mM) decreased glucose and Cro 2-induced insulin secretion. These results suggested that Na⁺ channel are involved in the insulin secretion. In this article, we also purified some peptide fragment from the treatment of reduced and carboxymethylated Cro 2 (RC-Cro 2) with cyanogen bromide and protease V8 from Staphylococcus aureus. The isolated pancreatic -cells were then treated with peptides only at high glucose concentration (16.7 mM), in this condition only two peptides induced insulin secretion. The amino acid sequence homology analysis of the whole crotamine as well as the biologically-active peptide allowed determining the consensus region of the biologically-active crotamine responsible for insulin secretion was KGGHCFPKE and DCRWKWKCCKKGSG.     

Growth of an adherent continuous cell line entrapped in a peg/alginate matrix

Summary CHO-K1 cells, an anchorage-dependent line, were entrapped in beads prepared from a Na alginate/polyethylene glycol mixture and grown, through successive passages, to an average maximum density of 4.5×10⁷ viable cells/g of bead. Cell growth and viability was unaffected by repeated alginate re-solubilization and reformation of the gel beads through five passages.     

Electrical coupling between cells in islets of langerhans from mouse

Summary Two microelectrodes have been used to measure membrane potentials simultaneously in pairs of mouse pancreatic islet cells. In the presence of glucose at concentrations between 5.6 and 22.2mm, injection of currenti into cell 1 caused a membrane potential change in this cell,V ₁, and, provided the second microelectrode was less than 35 m away, in a second impaled cell 2,V ₂. This result establishes that there is electrical coupling between islet cells and suggests that the space constant of the coupling ratio within the islet tissue is of the order of a few -cell diameters. The current-membrane potential curvesi-V ₁ andi-V ₂ are very similar. By exchange of the roles of the microelectrodes, no evidence of rectification of the current through the intercellular pathways was found. Removal of glucose caused a rapid decrease in the coupling ratioV ₂ /V ₁ . In steady-state conditions, the coupling ratio increases with the concentration of glucose in the range from 0 up to 22mm. Values of the equivalent resistance of the junctional and nonjunctional membranes have been estimated and found to change with the concentration of glucose. Externally applied mitochondrial blockers induced a moderate increase in the junctional resistance possibly mediated by an increase in intracellular Ca²⁺.     

Pulse schemes for the measurement of3 JC′Cγ and3 JNCγ scalar couplings in 15N,13C uniformly labeled proteins

Pulse sequences are presented for the measurement of³J_CC and³J_NC scalar couplings for allC containing residues in¹⁵N,¹³C uniformly labeled proteins. The methodsdescribed are based on quantitative J correlation spectroscopy pioneered byBax and co-workers [Bax et al. (1994) Methods Enzymol., 239, 79–105].The combination of ³J_CC and³J_NC scalar coupling constants allows theassignment of discrete rotameric states about the ₁ torsion angle in cases where such states exist or, alternatively,facilitates the establishment of noncanonical ₁conformations or the presence of rotameric averaging. The methods areapplied to a 1.5 mM sample of staphylococcal nuclease.     

Bioconversion of gitoxigenin by immobilized plant cells in a column bioreactor

Summary Daucus carota cells immobilized in a Ca-alginate performed the bioconversion of gitoxigenin to 5-hydroxygitoxigenin in a column bioreactor. The smooth spherical shape of the alginate beads was preserved for more than three weeks. The bioreactor was functional for more than thirty days as detected by the bioconversion activity. The rate of bioconversion was influenced by means of aeration.Issued as National Research Council of Canada Publication Number 19556     

The determination of alpha-adrenergic receptor concentration on rat pancreatic islet cells

The selective a2 adrenergic antagonist yohimbine has been shown to prevent the noradrenaline induced inhibition of insulin secretion from isolated rat islets of Langerhans, Binding studies utilizing [³H]yohimbine showed specific binding to dispersed rat islet cells with a K_d of 2.9 nM and receptor concentration of 645 fmols/mg protein. The use of chloroquine to inhibit receptor recycling did not affect binding of the ligand. Binding studies and secretion data are consistent with the suggestion that adrenergic receptors of the ₂ sub-type may play a dominant role in the regulation of insulin secretion.     

Calcitonin gene-related peptide and islet amyloid polypeptide stimulate insulin secretion in RINm5F cells through a common receptor coupled to a generation of cAMP

The question as to whether the homologous peptides CGRP and IAPP can regulate insulin secretion in RINm5F cells was addressed. Chicken CGRP displayed a reproducible inhibitory effect on insulin secretion within 0.1 and 1 nM concentrations and a stimulatory effect at higher concentrations. The maximal stimulatory effects on insulin secretion were obtained with 1.0 M of chicken CGRP (cCGRP), human -CGRP (h -CGRP) and human IAPP (hIAPP) which caused 246 ± 22, 302 ± 63 and 224 ± 14 percent increases of control levels, respectively (p < 0.001). Similarly, maximal accumulations of cAMP were obtained with 1.0 M of cCGRP, h -CGRP and hIAPP with the respective percent increases of control levels of 587 ± 24, 436 ± 41 and 410 ± 25 (p < 0.005). Thus the stimulatory effects on insulin secretion in RINm5F cells by cCGRP, h -CGRP and hIAPP appear to be mediated by the cAMP pathway. Chicken CGRP, the most potent peptide tested, displayed a correlated dose response stimulation of intracellular cAMP and insulin release within the concentration range of 10–1000nM. The EC₅₀ values of cCGRP for cAMP accumulation and insulin release were similar (20nM and 10 nM respectively). The stimulatory effect of IAPP on cAMP was not additive with that of cCGRP suggesting that IAPP action was mediated by CGRP receptors. This hypothesis was further sustained by a preferential inhibition of¹²⁵I[His]h -CGRP binding to RINm5F cells by cCGRP as compared to IAPP.We conclude that CGRP and IAPP, through a direct action on a chicken CGRP preferring receptor present in cells, stimulated insulin by a cAMP mediated pathway.